Approach

NAD⁺ is a fundamental molecule in human life and health as it participates in energy metabolism, cell signalling, mitochondrial homeostasis, and in dictating cell survival or death. Emerging evidence from preclinical and human studies indicates an age-dependent reduction of cellular NAD⁺, possibly due to reduced synthesis and increased consumption.

In preclinical models, NAD⁺ repletion extends healthspan and / or lifespan and mitigates several conditions, such as premature ageing diseases and neurodegenerative diseases. These findings suggest that NAD⁺replenishment through NAD⁺ precursors has great potential as a therapeutic target for ageing and age-predisposed diseases, such as Alzheimer’s disease. Here, we provide an updated review on the biological activity, safety, and possible side effects of NAD⁺ precursors in preclinical and clinical studies.

Major NAD⁺ precursors focused on by this review are nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), and the new discovered dihydronicotinamide riboside (NRH). In summary, NAD⁺ precursors have an exciting therapeutic potential for ageing, metabolic and neurodegenerative diseases.

Nicotinamide adenine dinucleotide (oxidised form, NAD⁺), first discovered as a coenzyme in fermentation in 1906, has been extensively studied throughout the last century with a total of four Nobel Prizes given to NAD/NADP-related discoveries (Fang et al., 2017; Harden and Young, 1906). Our understandings of the functions of NAD⁺ extend from a role in redox homeostasis, to its action as a fundamental metabolite participating in glycolysis, the tricarboxylic acid cycle (TCA) and mitochondrial oxidative phosphorylation (OXPHOS), to its participation in cell signalling pathways (Chalkiadaki and Guarente, 2015; Fang et al., 2017; Mouchiroud et al., 2013a; Verdin, 2015a). From a biosynthetic point of view, major known NAD⁺precursors are nicotinic acid (NA), nicotinamide (NAM), nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), and dihydronicotinamide riboside (NRH) (Bogan and Brenner, 2008; Katsyuba et al., 2020; Verdin, 2015a). Nicotinic acid (also named Vitamin B3 or niacin) has 2 other forms, (nicotinamide/NAM, also named niacinamide) and inositol hexanicotinate, which have different effects from nicotinic acid.

For many years, NAD⁺ precursors, such as NA and NAM, were researched and utilised in treatment of pellagra, a disease caused by a vitamin B3 deficiency that raged across Europe and the USA (Bogan and Brenner, 2008; Sydenstricker, 1958). As the knowledge of NAD⁺ and its role in metabolism and redox homeostasis expanded, NAD⁺ and its precursors gradually became more intensively studied in modern medicine, especially in the ageing field. Throughout the past two decades, NAD⁺ boosting molecules such as NA, NAM, NR, and NMN have displayed therapeutic potential in preventing ageing phenotypes and promoting healthy longevity (Canto et al., 2015; Lautrup et al., 2019). Rigorous trials of these NAD⁺boosters are required in order to determine the safety and efficacy of these molecules for ageing and different diseases. This review will discuss the current state of the literature surrounding cellular metabolism, functions, and possible therapeutic applications of NAD⁺ precursors.

In this section, we provide a summary of different known NAD⁺ synthetic pathways. Within the cell, NAD⁺ is reversibly converted to NADH. It also converts to NADP⁺which can be reversibly converted to NADPH. Furthermore, intracellular NAD⁺ is constantly consumed/degraded by enzymes such as sirtuins (SIRTs), ADP ribosyl transferases (ARTs) and poly (ADP-ribose) polymerases (PARPs), with NAMgenerated as its by-product (Fang et al., 2017). Thus, it is necessary to have efficient NAD⁺ synthesis in order to maintain a cellular NAD⁺ pool. This is achieved by a several synthesis pathways: the kynurenine pathway (de novo), the Preiss-Handler pathway, the salvage pathway, and the emerging NRH-salvage pathway. While the salvage pathway and the NRH-salvage pathway are intertwined, for clarity, we separate the two here (Fig. 1). The complexity of NAD⁺ biosynthesis provides multiple entry points for NAD⁺ augmentation, which lends itself to the potential for implementation of multiple pharmacological approaches through the administration of different precursors.

Genetic approaches, such as ubiquitous or tissue-specific knockout of PARP1 and CD38, and pharmacological methods, such as the use of NR, NMN, NAM, increase cellular NAD⁺ and exhibit broad health benefits. The in vitro restoration of NAD⁺mitigates bioenergetic impairment, improves mitochondrial biogenesis, restores the cellular protective capabilities against ROS, reduces DNA damage, promotes DNA repair, stimulates neuronal regeneration, as well as prohibits cellular senescence and improves stemness; the evidences were summarized elsewhere (Covarrubias et al., 2021; Fang et al., 2017). NAD⁺ augmentation also ameliorates systemic and organic dysfunction and improves healthspan and/or lifespan in the preclinical models, such as roundworm Caenorhabditis elegans, fruit fly Drosophila melanogaster, and mice (Covarrubias et al., 2020b; Fang et al., 2016a, 2014; Gomes et al., 2013; Mouchiroud et al., 2013b; Vannini et al., 2019; Wiley et al., 2016). Compared with genetic approaches to knockout NAD⁺-consuming enzymes to achieve cellular NAD⁺ preservation, practically pharmacological supplementation of NAD⁺precursors are easy to achieve (Fig. 2A). In view of the broad health benefits of NAD⁺precursors, there are more than 30 clinical trials on the use of NR or NMN currently listed on the NIH Clinical Trials database (clinicaltrials.gov). Many of the Phase I clinical trials are focused on bioavailability and safety (see Table 1). Further information outlining the proposed effects of NAD⁺ augmentation is described in Fig. 2B-C. Here, we will elaborate in detail on two topics: NAD + precursors in healthspan and longevity, and safety and potential side effects.